The long-term objective of these studies is to develop an effective vaccine against HIV. In the next funding period the main aim is to build on our finding that prime/boost immunization protocols with Gag and Env DNA based vaccines followed by L. monocytogenes, provide better immunity than does either vaccine protocol alone. The focus is to develop these vaccines for clinical trials. To achieve this there are three specific aims. In Specific Aim 1, attenuated mutants of L. monocytogenes suitable for vaccine use will be developed. One of these mutants is based on a totally avirulent D-alanine auxotroph complemented with a plasmid bearing the alanine racemase expressed by a weak promoter. This partially complements the strain so that it can replicate sufficiently to induce good immunity. The second type of mutant is unable to transport small peptides and cannot synthesize threonine so that it is attenuated by virtue of threonine starvation in vivo. Both mutants are 10 times less virulent than wild type Listeria in mice. The third mutant lacks two virulence factors required for cell-to-cell spread. In Specific Aim 2, the induction of mucosal immunity by the prime/boost vaccine approach will be measured using HIV Gag as a model antigen expressed by the attenuated strains. The generation and persistence of cellular immune responses induced by the prime/boost approach will be measured in GALT and in the spleen. Because of the difficulty in measuring such parameters in the primate model these studies will be performed in mice. In Specific Aim 3, the attenuated strains that have proved effective at inducing immunity in the mouse will be tested in the rhesus macaque/SHIV 89.6P model using the prime boost protocol with DNA based vaccines and oral L. monocytogenes. The addition of cytokines IL-12 and IL-15, to enhance the priming with the DNA vaccines, and other SIV antigens RT and Nef will also be explored. These studies will start in year 2 of the proposal. The hypothesis of this aim is that the attenuated strains will as effective as wild-type Listeria in delivering SHIV antigens to the immune system.